Association between Melatonin Receptor 1A Gene and Expression of Reproductive Seasonality in Sheep

To determine whether a link exists between reproductive seasonality and the structure of the melatonin receptor 1A (MTNR1A) gene, the latter was studied in nonseasonal estrous breeds (Small Tail Han and Hu ewes) and seasonal estrous breeds (Dorset, Suffolk and German Mutton Merino ewes). A large fragment of the exon 2 of the MTNR1A gene was amplified and a uniform fragment of 824 bp was obtained in 239 ewes of five breeds. The 824 bp PCR product was digested with restriction endonucleases Mnl I and Rsa I, and checked for the presence of restriction sites. The presence (allele M) or absence (allele m) of an Mnl I site at base position 605 led to three genotypes MM (236 bp/236 bp), Mm (236 bp/303 bp) and mm (303 bp/303 bp) in five sheep breeds. The presence (allele R) or absence (allele r) of a Rsa I site at base position 604 led to three genotypes RR (267 bp/267 bp), Rr (267 bp/290 bp) and rr (290 bp/290 bp) in five sheep breeds. Frequencies of MM and RR genotypes were obviously higher, and frequencies of mm and rr genotypes were obviously lower in nonseasonal estrous sheep breeds than in seasonal estrous sheep breeds. Sequencing revealed four mutations (G453T, G612A, G706A, C891T) in mm genotype compared to MM genotype and one mutation (C606T) in rr genotype compared to RR genotype. For polymorphic Mnl I and Rsa I cleavage sites, the differences of genotype distributions were very highly significant (p<0.01) between Small Tail Han ewes and seasonal estrous sheep breeds. In each group, no significant difference (p>0.05) was detected. These results preliminarily showed an association between MM, RR genotypes and nonseasonal estrus in ewes and an association between mm, rr genotypes and seasonal estrus in ewes. (


INTRODUCTION
Melatonin synthesis by the pineal gland occurs only during the hours of darkness, and gates photoperiodic information in mammals.Melatonin regulates circadian rhythms and reproduction changes in seasonally reproductive mammals (Ortavant et al., 1985;Reppert et al., 1994;Weaver and Reppert, 1996;Barrett et al., 1997).The circadian effects of melatonin appear to be mediated by melatonin receptors in the hypothalamic suprachiasmatic nucleus, the site of a circadian clock (Weaver and Reppert, 1996), and the reproductive effects mediated by melatonin receptors in the hypophyseal pars tuberalis (Reppert et al., 1994).Annual fluctuations in timing and duration of the nocturnal elevation in circulating melatonin is known to be a key factor influencing seasonal reproduction in sheep (Malpaux et al., 1996).Melatonin exerts its reproductive and circadian effects through the binding to pharmacologically specific, high-affinity, G-protein-coupled receptors (Dubocovich and Takahashi, 1987;Reppert et al., 1988;Vanecek, 1988;Roca et al., 1996).Melatonin receptor 1a (MTNR1A), a high-affinity melatonin receptor that mediates these two major biological functions of melatonin in mammals had been cloned by Reppert et al. (1994).By microsatellite markers and two-point linkage analysis, Messer et al. (1997) mapped MTNR1A gene to ovine chromosome 26, between microsatellites CSSM43 and BM6526.Discovery by Messer et al. (1997) of two polymorphic RFLP sites within the ovine MTNR1A gene provided opportunity to evaluate the influence of this gene on seasonal reproduction.Pelletier et al. (2000), Ji et al. (2003), Chu et al. (2003) and Notter et al. (2003) reported that the homozygous genotype for the absence of a polymorphic Mnl I site at position 605 of exon 2 of MTNR1A gene was associated with seasonal anovulatory activity in ewes.
In most sheep breeds, ovulatory activity of ewes is generally inhibited for several consecutive months of the year, referred to as the anestrous season, which occurs in spring.However, both Small Tail Han sheep and Hu sheep, that are excellent local breeds in China, display significant characteristics of non-seasonal estrus (Tu, 1989).Small Tail Han ewes continue to cycle throughout the year (Zhang, 1995;Wang et al., 1997;Jia et al., 2005).The objectives of the present study were firstly to detect the PCR-RFLP polymorphism of MTNR1A gene in the non-seasonal estrous breeds (Small Tail Han and Hu ewes) and seasonal estrous breeds (Dorset, Suffolk and German Mutton Merino ewes), and secondly to investigate the associations between MTNR1A gene and reproductive seasonality in ewes.This achievement could provide a theoretical basis for genetically controlling the ovine estrus, and lay a foundation for both changing the ovine estrous seasonality and improving the reproductive performance by means of gene knockout.

Genomic DNA preparation
Jugular blood samples (10 ml per ewe) were collected from 137 Small Tail Han ewes (Jiaxiang Sheep Breeding Farm located in Jiaxiang County, Shandong Province, China), 30 Dorset, 24 Suffolk and 21 German Mutton Merino ewes (HITEK Ranch (Beijing) Ltd.Co. located in Dasungezhuang Town, Shunyi District, Beijing, China), and 27 Hu ewes (Yuhang Hu Sheep Breeding Farm located in Yuhang District, Hangzhou City, Zhejiang Province, China) using acid citrate dextrose as an anticoagulant.These ewes were chosen at random.Genomic sheep DNA was extracted from whole blood by phenol-chloroform method, and then dissolved in TE buffer (10 mmol/L Tris•Cl (pH 8.0), 1 mmol/L EDTA (pH 8.0)) and kept at -20°C.
PCR products of 7 µl were digested separately with 5 U Mnl I (New England Biolabs, Beverly, MA, USA) and 5 U Rsa I (Promega, Madison, WI, USA) at 37°C overnight.The resultant fragments were separated by electrophoresis on 8% polyacrylamide gels in parallel with a pBR322/Msp I marker.The gels were stained with silver nitrate (silver staining), photographed and analyzed using an AlphaImager TM 2200 and 1220 Documentation and Analysis Systems (Alpha Innotech Corporation, San Leandro, CA, USA).
Genotyping was performed in the 239 DNA samples from 137 Small Tail Han, 30 Dorset, 24 Suffolk, 21 German Mutton Merino and 27 Hu ewes.

Cloning and sequencing
After RFLP analysis, PCR products of different homozygous genotypes were separated on 1.5% agarose gels and recovered using Geneclean II kit (Promega, Madison, WI, USA).Each DNA fragment was ligated into the pGEM-T Easy vector (Promega, Madison, WI, USA) according to the manufacturer's instructions.The ligation reactions were carried out in 10 µl volume containing PCR product 1 µl, pGEM-T Easy vector (50 ng/µl) 1 µl, T 4 ligase (3 U/µl) 1 µl, 2×ligation buffer 5 µl, ddH 2 o 2 µl.Each DNA fragment was then transformed into Escherichia coli DH5α competence cell.Positive clones of transformed cells were identified by restriction enzyme digestion.Two clones of each homozygous genotype were selected and sequenced.Each clone was sequenced for twice.The target DNA fragments in recombinant plasmids were sequenced from

Statistical analysis
Distribution of genotypes between different sheep breeds was analyzed using the chi-square method.

PCR amplification of exon 2 of ovine MTNR1A gene
In the present study, the primers for the exon 2 of ovine MTNR1A gene were used for amplification genomic DNA of different sheep breeds and the PCR products were separated on 1.5% agarose gels.The result showed that amplification fragment size 824 bp was consistent with the target one and had good specificity (Figure 1), which could be directly analyzed by RFLP.

RFLP analysis of exon 2 of ovine MTNR1A gene
There are seven cleavage sites (218 bp, 36 bp, 67 bp, 236 bp, 22 bp, 28 bp, 82 bp, 135 bp) for Mnl I within the amplification fragment, but only one was shown to be polymorphic (Chu et al., 2003).This site was at position 605 in the reference sequence (Reppert et al., 1994).Digestion with Mnl I yielded polymorphic fragments of 236 bp and 67 bp when the cleavage site was present (allele M) or a single 303 bp fragment if the cleavage site was absent (allele m).Three genotypes MM (236 bp/236 bp), Mm (236 bp/303 bp) and mm (303 bp/303 bp) were detected in five sheep breeds (Figure 2).
There are four cleavage sites (53 bp, 267 bp, 23 bp, 411 bp, 70 bp) for Rsa I within the amplification fragment, but only one was shown to be polymorphic (Chu et al., 2003).This site was at position 604 in the reference sequence (Reppert et al., 1994).Digestion with Rsa yielded Ⅰ polymorphic fragments of 267 bp and 23 bp when the cleavage site was present (allele R) or a single 290 bp fragment when the cleavage site was absent (allele r).Three genotypes RR (267 bp/267 bp), Rr (267 bp/290 bp) and rr (290 bp/290 bp) were detected in five sheep breeds (Figure 3).

Sequencing of different homozygous genotypes
Sequencing verified the presence (or absence) of the polymorphic Mnl I and Rsa I cleavage sites as assessed by polyacrylamide gel electrophoresis.The sequencing results indicated that genotype MM had the same sequence as that of U14109 in GenBank and was referred to as wild type.Genotype mm had four mutations(G453T, G612A, G706A, C891T)compared to MM genotype and was referred to as mutation genotype.Mutation at position 706 resulted in the substitution of a valine by an isoleucine in the amino acid sequence.The other mutations were silent.Genotype RR had the same sequence as that of U14109 in GenBank and was referred to as wild type.Genotype rr had one mutation (C606T) compared to RR genotype and was referred to as mutation genotype.Mutation at position 606 was silent.In all, a total of 5 different mutations were registered (see Table 1, Figures 4 and 5).

Allele and genotype frequencies of MTNR1A gene in different sheep breeds
Allele and genotype frequencies of MTNR1A gene in five sheep breeds were presented in Table 2.
Frequencies of MM and RR genotypes were obviously higher, and frequencies of mm and rr genotypes were obviously lower in nonseasonal estrous sheep breeds than in seasonal estrous sheep breeds.These results preliminarily showed an association between MM, RR genotypes and nonseasonal estrus in ewes, an association between mm, rr genotypes and seasonal estrus in ewes.

Test of difference for MTNR1A genotype distribution in different sheep breeds
The test result of difference for MTNR1A genotype distribution in five sheep breeds was summarized in Table 3.
For polymorphic Mnl I and Rsa I cleavage sites, the differences of genotype distributions were very highly

Polymorphism of ovine MTNR1A gene
The 824 bp product of exon 2 of the ovine MTNR1A gene was digested with restriction endonucleases Mnl I and Rsa I in sheep populations.For Mnl I site, the 286 bp and 236 bp fragments were polymorphic (Messer et al., 1997;Notter et al., 2003), the 303 bp and 236 bp fragments were polymorphic (Pelletier et al., 2000;Chu et al., 2003;Ji et al., 2003).For Rsa I site, the 295 bp and 290 bp fragments were polymorphic (Messer et al., 1997;Notter et al., 2003), the 290 bp and 267 bp fragments were polymorphic (Chu et al., 2003;Ji et al., 2003).The results of this study were consistent with those of Pelletier et al. (2000), Ji et al. (2003) and Chu et al. (2003).
Functional differences in MTNR1A were not anticipated for different genotypes.This study and Pelletier et al. (2000) demonstrated that mutation at position 612, responsible for the absence of Mnl I site, was always found to be associated with the 3 other mutations at positions 453, 706, and 891. This study and Pelletier et al. (2000) revealed that the mutation at position 706 leaded to the substitution of a valine at position 220 by an isoleucine in the fifth transmembrane domain.Moreover, isoleucine 220 is close to histidine 211, whose mutation modifies the K d value of 125 I-melatonin binding to Mel 1αβ receptor (Conway et al., 1997).The importance of this particular amino acid merits further study by mutagenesis.

Reproductive seasonality and ovine MTNR1A gene
Spontaneous ovulation in early spring in the Merinos d'Arles ewes had been studied by Hanocq et al. (1999), and this trait was found to have a significant heritability (h 2 = 0.20) in a model taking into account several physiological parameters such as weight and age of animals.Montgomery and Kawker (1987) and Hanocq et al. (1999) suggested the existence of a common overall factor involved in the control of reproductive seasonality.One possible candidate could be MTNR1A gene.Pelletier et al. (2000), Ji et al. (2003), Chu et al. (2003) and Notter et al. (2003) reported that the homozygous genotype for the absence of a polymorphic Mnl I site at position 605 of exon 2 of MTNR1A gene was associated with seasonal anovulatory activity in ewes.This study showed an association between MM genotype and nonseasonal estrus in ewes, an association between mm genotype and seasonal estrus in ewes.The next step in the present study will be to establish whether the association between genotype and ovarian seasonality is a true genetic linkage.The fact that this association remained within families supported this hypothesis (Pelletier et al., 2000).
This study showed an association between RR genotype and nonseasonal estrus in ewes, an association between rr genotype and seasonal estrus in ewes.The next step in the present study will be to establish whether the association between genotype and ovarian seasonality is a true genetic linkage.

Figure 4 .Figure 3 .
Figure 4. Sequence comparison of MM and mm genotypes of MTNR1A gene in sheep.

Table 1 .
Positions of mutations and base and amino acid changes in exon 2 of MTNR1A gene in five sheep breeds a Base or amino acid positions corresponding to U14109 of GenBank.

Table 2 .
Allele and genotype frequencies of PCR-RFLP of the MTNR1A gene in five sheep breeds Sequence comparison of RR and rr genotypes of MTNR1A gene in sheep.